PPARs: Interference with Warburg' Effect and Clinical Anticancer Trials

Abstract

The metabolic/cell signaling basis of Warburg's effect ("aerobic glycolysis") and the general metabolic phenotype adopted by cancer cells are first reviewed. Several bypasses are adopted to provide a panoramic integrated view of tumoral metabolism, by attributing a central signaling role to hypoxia-induced factor (HIF-1) in the expression of aerobic glycolysis. The cancer metabolic phenotype also results from alterations of other routes involving ras, myc, p53, and Akt signaling and the propensity of cancer cells to develop signaling aberrances (notably aberrant surface receptor expression) which, when present, offer unique opportunities for therapeutic interventions. The rationale for various emerging strategies for cancer treatment is presented along with mechanisms by which PPAR ligands might interfere directly with tumoral metabolism and promote anticancer activity. Clinical trials using PPAR ligands are reviewed and followed by concluding remarks and perspectives for future studies. A therapeutic need to associate PPAR ligands with other anticancer agents is perhaps an important lesson to be learned from the results of the clinical trials conducted to date.

Figure 1

Metabolism of glycolysis-derived NADH and pyruvate in normoxia (a), anoxia and cancer (b). (a) Normoxic normal cells classically oxidize glucose to completion. Cytosolic enzymes convert 1 molecule of glucose to 2 molecules of pyuvate and along with 2 ATP and 2 NADH. Mitochondrial oxidations of glucose-derived pyruvate and NADH involve pyruvate dehydrogenase (PDH), Krebs cycle (KC), and respiratory chain electron transfer/oxidative phosphorylation (OXPHOS) complexes I, II, II, IV, and V, yielding classically 34 ATP. Complete oxidation of glucose therefore results in the production of 36 (2 cytosolic + 34 mitochondrial) ATP. (b) The contribution of mitochondria to glucose oxidation is disrupted in anoxic normal or cancer cells by the arrest of mitochondrial respiration (lack of oxygen in anoxia) and in normoxic and anoxic cancer cells by different convergent mechanisms. Among these, reduced pyruvate dehydrogenase activity may result from overexpressed pyruvate dehydrogenase kinase 1 and limited access of pyruvate to the mitochondria due to the closed state of mitochondrial outer membrane voltage-dependent anionic channel (VDAC). Reduced activities of the respiratory chain complexes I and IV and muted Krebs cycle enzymes may be also encountered. Pyruvate, formed intracytosolically from glucose via glycolysis, is no longer oxidized in mitochondria and is metabolized by cytosolic lactate dehydrogenase. In cancer cells, it must be stressed that the metabolic events mentioned above take place in the context of fuel producing glycolysis in which cytosolic net ATP formation occurs. In turn, the glycolytic flux may be blocked at the pyruvate kinase step (see Figure 4), resulting in biosynthetic precursor-producing glycolysis with little or no net glycolytic ATP or pyruvate production. Cytosolic pyruvate is then provided via other routes (precursors other than glucose) including serinolysis and glutaminolysis, pathways which refer to conversions of serine and glutamine to lactate, respectively.

Figure 2

Activation and inactivation pathways for hypoxia-induced factor 1 (HIF-1) in normoxia, hypoxia/anoxia, and cancer. (a) In normoxic normal cells, molecular oxygen (O₂) sensing by membrane NADPH oxidase results in formation of superoxide radical anions and subsequently in the control of the HIF-1 signaling pathway via proteolytic degradation of the HIF-1α subunit. These events prevent the recruitment of functional HIF-1 which results from heterodimerization of HIF-1α and β subunits. In anoxia/hypoxia, a severe drop in the levels of NADPH oxidase-driven superoxide radical anion prevents oxidative and proteolytic damage of the HIF-1α subunit which then becomes available to form functional HIF-1 and results in signaling activation. In cancer cells, the HIF-1 signaling pathway may be overexpressed via, for instance, mechanisms illustrated in Panel b. (b) This panel details hydroxylation and the subsequent steps of the HIF-1 inactivation pathway and its disruption in cancer cells. Mechanisms that neutralize the HIF-1 pathway in cancer cells may include alteration of gene expression for succinate dehydrogenase and fumarase (the net results of which are a rise in succinate levels which interfere with the hydroxylation steps by product inhibition) (mechanism 1) and for von Hippel-Lindau protein (mechanism 2) (preventing formation of E3-ubiquitin ligase proteic complex and hence HIF-1α inactivation). Other comments are in the text.

Figure 3

Illustration of how biased HIF-1 signaling in cancer cells may cause Warburg' effect and lead to modified protein content and subcellular localization (a), metabolism (b), and cancer-specific therapeutic opportunities (c). The role of aberrant HIF-1 signalling in cancer cells by encoding proteins (a) which modify the intermediary metabolism in a way that favors the emergence of glycolytic metabolism even in normoxic conditions (b). Note the convergence of the HIF-1-driven increase of proteins, convergence which favors tumor vascularization (Figure 2(a)), and aerobic glycolysis (this figure). Panel (c) illustrates therapeutic opportunities related to cancer cell metabolism. Inhibition of glucose transport and activation disrupts Warburg's effect, depriving cancer cells of their preferential metabolic substrate. Inhibition of hexokinase may in addition lead to its detachment from mitochondrial VDAC. Blocking the utilization of glucose 6-phosphate by increasing its concentrations secondarily leads to hexokinase inhibition and hence its detachment from VDAC (reopening this channel). Direct interaction with VDAC might also disrupt the closed state of the channel. The closed channel may be, however, bypassed by small permeant compounds such as pyruvate methyl ester (pme) and dichloroacetate (dca). When VDAC is closed, pyruvate methyl ester, in contrast to pyruvate, can enter the mitochondrial matrix where an esterase produces pyruvate, following this the action of residual pyruvate dehydrogenase generates an electron flux towards the respiratory chain (at the level of complex I), a feature capable of triggering mitochondrial apoptosis. Dichloroacetate inhibits pyruvate dehydrogenase kinase activity and then restores substantial pyruvate dehydrogenase activity and subsequent flux towards respiratory chain. Lipopphilic cationic compounds are attracted by cancer cell mitochondria which present abnormally high negative electric charges consequently to reduced electron chain proton efflux and to the closed state of VDAC (resulting in accumulation of small negative metabolites trapped within the mitochondria). Lipohilic cations may cross mitochondrial membranes, bypassing VDAC and being insensitive to the closed state of this channel. The intramitochondrial accumulation of lipophilic cations induces destruction and depletion of mitochondrial DNA. Abbreviations are GLUT1, glucose transporter 1; LDHA, lactate dehydrogenase A; MCT4, monocarboxylate transporter 4; PDH, pyruvate dehydrogenase; PDK, pyruvate dehydrogenase kinase1; HKII, hexokinase II; VDAC, voltage-dependent anionic channel; pme, pyruvic methyl ester; dca, dichloracetate; RC, respiratory chain.

Figure 4

Tetrameric M2 pyruvate kinase-driven fuel-generating (green panel) and dimeric M2 pyruvate kinase-driven biosynthetic precursor-generating (red panels) glycolysis (yellow panel) in cancer cells. Tumoral M2 pyruvate kinase exists in a dimeric inactive form that blocks pyruvate and ATP formation from glucose. It induces the accumulation of energy-rich phosphometabolites found upstream in the glycolytic pathway and off which biosynthetic processes may branch. Interconversion to the active tetrameric form of the enzyme may occur when the glucose supply is high, leading to a rise in fructose 1,6 bisphosphate which stimulates this tetrameric conversion. When glucose levels are high, cancer cells may then produce energy at the same time as supplying biosynthetic pathways. In this situation, the rise in glycolytic intermediary rich energy phosphometabolites results not from a block located downstream of their production but from an increased load of glycolysis by glucose. When glucose levels fall again, the subsequent decrease in fructose 1,6 bisphosphate results in recruitment of the dimeric inactive form of M2 pyruvate kinase. In this case, both energy and pyruvate production (and hence formation of lactate) by the tumor may derive from the catabolism of aminoacids such as glutamine and serine. The latter sets of metabolic reactions, by analogy with glycolysis for glucose to lactate production, are referred to as glutaminolysis and serinolysis, respectively. The supply of these aminoacids to the tumor is associated at distance with notably muscle proteolysis, explaining the progression of patients towards a cachectic state when the tumor gains in growth and development. Cachexy may be also favored by energy wasting associated to uncoupling of mitochondria in some cancer cell lines. Except with tumors such as insulinoma and hepatoma, for instance, no hypoglycaemia is, however, induced in patients since a sustained production of lactate is ensured by the tumor from these aminioacids. Lactate may be recycled to glucose by gluconeoformator cells, mainly hepatocytes (Cori's cycle). Biosynthetic pathways branching off glycolysis include sialic acid, nucleic acid, aminoacid, ether glycerolipid, and ester glycerolipid anabolic pathways. The latter pathway is not emphasized here by inclusion in a red panel because in many cancer cell lines glycerol phosphate dehydrogenase is deficient thus reducing the availability of glycerol 3-phosphate and limiting incorporation of neoformed fatty acids into lipids. Fatty acid synthase is often overexpressed, and, along with the removal of fatty acids (known for immunosuppressive properties) outside the cell, it allows tumoral cells to cope with the massive rise in glycolysis-driven NADH and proton (H⁺) formation (glyceraldehyde 3-phosphate dehydrogenase step), avoiding excess acidification and consequent cell death. The threshold for reversible interconversion of tetrameric to dimeric M2 pyruvate kinase may be lowered by oncogenes in favor of the dimeric form. As mentioned in the text, the tetrameric active form is part of the glycolytic complex (a complex which groups most glycolytic enzymes for optimal metabolic function and energy production) whereas the dimeric form separates from this glycolytic complex.

Figure 5

Modulation of gene expression by PPARs and inflammatory transcription factors: transactivation and transrepression mechanisms. The activation of PPARs (a) and inflammatory transcription factors (b) modulates gene expression via specific response elements (RE), resulting mainly in the upregulation of proteins involved in several metabolic and inflammatory pathways, respectively. The two routes may connect through protein-protein interaction upstream to DNA-protein interaction (c). This transrepressive action of a pathway on the other is a basis for the anti-inflammatory/antioxidative properties of PPARs, and though less often mentioned, for the anti-PPAR properties of inflammatory/oxidative processes. Abbreviations: COX2, cyclooxygenase 2; ICAM, inducible cell adhesion molecule; PPRE, PPAR-responsive element; RXRα, retinoid acid X receptor α. The mechanisms by which metabolic changes and anti-inflammatory properties induced by PPARs interfere with the Warburg's effect and cancer development are considered in the text.

Figure 6

Arachidonic acid metabolism as a provider of physiological PPAR ligands. The figure illustrates in a nonexhaustive way that arachidonic acid (AA) may generate various lipoxygenase (LOX) (hydroxyeicosatrienes, HETEs; hydroperoxyeicosatrienes; HPETEs, leucotrienes, LTs) and cyclooxygenase- (COX) (prostaglandins, PGs; thromboxanes, TXs) derived metabolites. Among these metabolites, LTB4, prostacyclin (PGI2), and 15-deoxy PGJ2 (PGJ2) represent ligands of each of the three PPAR isoforms, PPARα, β, and γ, respectively. Subsequent physiological activation of PPARs impacts the formation of such ligands via a negative feedback loop by increasing their degradation and lowering their formation. Indeed, PPARα activation stimulates microsomal ω-hydroxylase (in humans) and peroxisomal β-oxidation (in rodents, for instance but not in humans) and hence degradation of each of the precited PPAR ligands. Each of the PPARs may upregulate the activity of antioxidant enzymes and downregulate inflammatory proteins. Preventing induction of cyclooxygenase type 2 is a way to counteract inflammation-driven increase in arachidonic metabolites which are cyclooxygenase-dependent ligands of PPARs. The interest of a PPAR-based therapy in preventing the synergism between stromal cell inflammation and tumor development/invasion is explained in the text.

Figure 7

Potential anticancer value of metabolic changes interfering with Warburg's effect and mediated essentially by PPARα (the metabolic events numbered in the figure are explained in the text).

Figure 8

PPARs, metabolic syndrome and cancer. This figure illustrates that (i) PPAR activation may counteract the development of metabolic syndrome, (ii) metabolic syndrome increases the risk for cancer, and (iii) PPARs and their ligands exhibit pro-and antitumoral properties towards cancer progression. Metabolic syndrome is considered as a procancer target antagonised by PPARs, a feature (not further discussed in the text) giving support to the view of a possible preventive anticancer action of PPAR ligands which may be supplied by the diets (polyunsaturated fatty acids, for instance). The figure also compares the links leading to the current medical and pharmacological applications of PPAR ligands.